Method for Producing Peptide Ace Inhibitors

ABSTRACT

A method for producing ACE Inhibitor peptides from a protein source or plasma is disclosed. The method utilizes proteolysis by intestinal, blood-circulating, or membrane-bound proteases. The initial synthesis step could require obtaining a protein source either from a human or animal. A protease is added to either a given plasma protein or plasma and incubated. Following incubation, the protease activity must be quenched using a protease inhibitor to inactivate the protease. After incubation with protease inhibitor, the solution will contain a mixture of bioactive ACE inhibitory peptides and inert peptides. This mixture may be purified to select for the ACE inhibitory peptides through centrifugation. The mixture may also be sterilized to remove any microbial contaminants. The ACE inhibitory peptides can be mixed with protein powders, incorporated into baked good and put into other food products to provide food products with the added benefit of lowering blood pressure.

CROSS-REFERENCE TO PROVISIONAL APPLICATION

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application Ser. No. 62/634,451, entitled “METHOD FORPRODUCING PEPTIDE ACE INHIBITORS FROM PLASMA AND PROTEIN SOURCES,” whichwas filed on Feb. 23, 2018, the disclosure of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

Embodiments are generally related to peptide angiotensin-convertingenzyme (ACE) inhibitors. Embodiments are additionally related to amethod for producing peptide ACE inhibitors. Embodiments also relate toproducing peptide ACE inhibitors from plasma and protein sources.

BACKGROUND

Hypertension is a condition afflicting millions of people per year. Itis a co-morbidity related to diabetes, atherosclerosis and a host ofother conditions. Hypertension results when angiotensin II causes themuscles surrounding blood vessels to contract and narrow the vessels.Blood pressure increases when the vessels narrow.

A typical treatment for hypertension is angiotensin converting enzyme(ACE) inhibitors to maintain lower blood pressure. ACE inhibitorsfunction by inhibiting the enzyme that converts angiotensin I toangiotensin II. Angiotensin converting enzyme conversion of angiotensinIto angiotensin II physiologically results in vasoconstriction of theblood vessels, causes an increase in blood pressure, and often resultsin kidney disease. ACE inhibitors help to prevent this type of kidneydisease by reducing intraglomerular pressure caused by release ofangiotensin II.

Previous studies show that ACE inhibitors have limitations in terms ofhow effective they can be in inhibiting the functionality of therenin-angiotensin-aldosterone system (RAAS). The renin-angiotensinsystem, working together with the kidneys, is the body's most importantlong-term blood pressure regulation system. This can result in adecreased state of health for patients as well as money spent by thepatient on drugs that do not fully achieve their designed purpose. Inaddition, methods used to produce ACE inhibitors have historically beencostly and time-intensive.

Accordingly, the composition and method disclosed herein is intended tosolve such problems such that peptide ACE inhibitors for reducing bloodpressure are produced from plasma and protein sources in a costefficient manner.

BRIEF SUMMARY

The following summary is provided to facilitate an understanding of someof the innovative features unique to the embodiments disclosed and isnot intended to be a full description. A full appreciation of thevarious aspects of the embodiments can be gained by taking the entirespecification, claims, drawings, and abstract as a whole.

It is, therefore, an aspect of the disclosed embodiments to provideenhanced peptide angiotensin-converting enzyme (ACE) inhibitors.

It is another aspect of the invention to provide a method for producingpeptide ACE inhibitors.

It is yet another aspect of the disclosed invention to produce peptideACE inhibitors from plasma and protein sources.

The aforementioned aspects and other objectives and advantages can nowbe achieved as described herein. A method for producing ACE Inhibitorpeptides from a protein source or plasma is disclosed. The methodutilizes proteolysis by intestinal, blood-circulating, or membrane-boundproteases. The initial synthesis step could require obtaining a proteinsource either from a human or animal. A protease is added to either agiven plasma protein or plasma and incubated. Following incubation, theprotease activity must be quenched using a protease inhibitor toinactivate the protease. After incubation with protease inhibitor, thesolution will contain a mixture of bioactive ACE inhibitory peptides andinert peptides. This mixture may be purified to select for the ACEinhibitory peptides through centrifugation. The mixture may also besterilized to remove any microbial contaminants. The ACE inhibitorypeptides can be mixed with protein powders, incorporated into baked goodand put into other food products to provide food products with the addedbenefit of lowering blood pressure.

In an embodiment, a method of making polypeptide angiotensin convertingenzyme (ACE) inhibitors from plasma proteins is disclosed. In certainembodiments, the method can comprise mixing a protein source with atleast one protease; quenching the at least one protease mixed with theprotein source to create the polypeptide angiotensin converting enzyme(ACE) inhibitors; and inhibiting the at least one protease mixed withthe protein source completely with the polypeptide angiotensinconverting enzyme (ACE) inhibitors.

In another embodiment, the protein source is mixed with the at least oneprotease for up to 24 hours at up to 40 degrees Celsius. In anembodiment, the protein source is selected from at least one of serumalbumin and macroglobulin. In yet another embodiment, the protein sourceis at a concentration up to a saturation point of the protein source. Inanother embodiment, the at least one protease is selected at least oneof trypsin, chymotrypsin, and elastase. In some embodiments, the atleast one protease is at a concentration up to one tenth of aconcentration of a plasma protein. In yet another embodiment, quenchingcomprises removing protease activity by any chemical or mechanical meanscomprising at least one of filtration, boiling, or utilizing a proteaseinhibitor, wherein the protease inhibitor is at a concentration up toone tenth of a concentration of the at least one protease. In certainembodiments, the method can further comprise incorporating thepolypeptide angiotensin converting enzyme (ACE) inhibitors into foodproducts, wherein the food products can comprise at least one of proteinpowder and a baked good and a user consuming the food productscontaining the polypeptide angiotensin converting enzyme (ACE)inhibitors to lower the blood pressure of the user.

In another embodiment, a method of making polypeptide angiotensinconverting enzyme (ACE) inhibitors from plasma proteins is disclosed. Incertain embodiments, the method can comprise: mixing plasma with atleast one protease; quenching the at least one protease mixed with theplasma to create the polypeptide angiotensin converting enzyme (ACE)inhibitors; and inhibiting the at least one protease mixed with theplasma completely with the polypeptide angiotensin converting enzyme(ACE) inhibitors.

In some embodiments, the plasma is mixed with the at least one proteasefor up to 24 hours at up to 40 degrees Celsius. In other embodiments,the at least one protease is selected from at least one of trypsin,chymotrypsin, and elastase. In yet another embodiment, the at least oneprotease is at a concentration up to 1 g/mL. In an embodiment, quenchingcomprises removing protease activity by any chemical or mechanical meanscomprising at least one of filtration, boiling, or utilizing a proteaseinhibitor, wherein the protease inhibitor is at a concentration up toone tenth of a concentration of the at least one protease. In yetanother embodiment, the method can further comprise incorporating thepolypeptide angiotensin converting enzyme (ACE) inhibitors into foodproducts, wherein the food products can comprise at least one of proteinpowder and a baked good and a user consuming the food productscontaining the polypeptide angiotensin converting enzyme (ACE)inhibitors to lower the blood pressure of the user.

In another embodiment, a method of making polypeptide angiotensinconverting enzyme (ACE) inhibitors from plasma proteins is disclosed. Insome embodiments, the method can comprise: mixing plasma with at leastone protease, wherein the at least one protease comprises chymotrypsin;quenching the at least one protease with the mixed plasma andchymotrypsin to create the polypeptide angiotensin converting enzyme(ACE) inhibitors; and inhibiting chymotrypsin mixed with the plasmacompletely with the polypeptide angiotensin converting enzyme (ACE)inhibitors.

In certain embodiments, the plasma is mixed with chymotrypsin for up to24 hours at up to 40 degrees Celsius. In other embodiments, chymotrypsinis at a concentration up to 1 g/mL. In yet another embodiment, quenchingcomprises removing protease activity by any chemical or mechanical meanscomprising at least one of filtration, boiling, or utilizing a proteaseinhibitor, wherein the protease inhibitor is at a concentration up toone tenth of a concentration of chymotrypsin. In some embodiments, themethod can further comprise incorporating the polypeptide angiotensinconverting enzyme (ACE) inhibitors into food products, wherein the foodproducts can comprise at least one of protein powder and a baked goodand a user consuming the food products containing the polypeptideangiotensin converting enzyme (ACE) inhibitors to lower the bloodpressure of the user.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, in which like reference numerals refer toidentical or functionally-similar elements throughout the separate viewsand which are incorporated in and form a part of the specification,further illustrate the present invention and, together with the detaileddescription of the invention, serve to explain the principles of thepresent invention.

FIG. 1 illustrates a schematic of the ACE inhibitor production method,which can be implemented in accordance with an example embodiment;

FIG. 2 illustrates a chart of the average values of animal bloodpressure following femoral vein and artery cannulation, in accordancewith an embodiment;

FIG. 3 illustrates a chart of the average values of animal bloodpressure following femoral vein and artery cannulation, in accordancewith an embodiment;

FIG. 4 illustrates a graph of concentrations of 1:500 Albumin toProtease ratio mixed with other compounds, in accordance with anembodiment, in accordance with an embodiment;

FIG. 5 illustrates a graph of concentrations of 1:1000 Albumin toProtease ratio mixed with other compounds, in accordance with anembodiment, in accordance with an embodiment;

FIG. 6 illustrates a graph comparing blood pressure after injection withTrypsin and Albumin, in accordance with an embodiment;

FIG. 7 illustrates a graph comparing blood pressure after injection withChymorypsin and Albumin, in accordance with an embodiment; and

FIG. 8 illustrates a graph comparing blood pressure after injection withChymorypsin and Plasma, in accordance with an embodiment.

DETAILED DESCRIPTION

The particular values and configurations discussed in these non-limitingexamples can be varied and are cited merely to illustrate one or moreembodiments and are not intended to limit the scope thereof.

Subject matter will now be described more fully hereinafter withreference to the accompanying drawings, which form a part hereof, andwhich show, by way of illustration, specific example embodiments.Subject matter may, however, be embodied in a variety of different formsand, therefore, covered or claimed subject matter is intended to beconstrued as not being limited to any example embodiments set forthherein; example embodiments are provided merely to be illustrative.Likewise, a reasonably broad scope for claimed or covered subject matteris intended. Among other things, for example, subject matter may beembodied as methods, devices, components, or systems. Accordingly,embodiments may, for example, take the form of hardware, software,firmware, or any combination thereof (other than software per se). Thefollowing detailed description is, therefore, not intended to beinterpreted in a limiting sense.

Throughout the specification and claims, terms may have nuanced meaningssuggested or implied in context beyond an explicitly stated meaning.Likewise, the phrase “in one embodiment” as used herein does notnecessarily refer to the same embodiment and the phrase “in anotherembodiment” as used herein does not necessarily refer to a differentembodiment. It is intended, for example, that claimed subject matterinclude combinations of example embodiments in whole or in part.

In general, terminology may be understood, at least in part, from usagein context. For example, terms such as “and”, “or”, or “and/or” as usedherein may include a variety of meanings that may depend, at least inpart, upon the context in which such terms are used. Typically, “or” ifused to associate a list, such as A, B, or C, is intended to mean A, B,and C, here used in the inclusive sense, as well as A, B, or C, hereused in the exclusive sense. In addition, the term one or more as usedherein, depending at least in part upon context, may be used to describeany feature, structure, or characteristic in a singular sense or may beused to describe combinations of features, structures, orcharacteristics in a plural sense. Similarly, terms such as “a”, “an”,or “the”, again, may be understood to convey a singular usage or toconvey a plural usage, depending at least in part upon context. Inaddition, the term “based on” may be understood as not necessarilyintended to convey an exclusive set of factors and may, instead, allowfor existence of additional factors not necessarily expressly described,again, depending at least in part on context.

In certain embodiments, a method for producing ACE Inhibitor peptidesfrom a protein source or plasma is disclosed. The disclosed embodimentsuse either a purified protein or plasma as a substrate to generatepeptides. Plasma is a low-cost alternative to using purified proteins asplasma may be obtained from animals with minimal expense.

The disclosed method utilizes proteolysis by intestinal,blood-circulating, or membrane-bound proteases. During physiologicalshock stemming from sepsis, hemorrhage, peritonitis, or other trauma,hypervolemia is experienced by the patient. The patient's body respondsto this state of hypervolemia and decreased blood pressure by pulling influid from the patient's organs and other tissues. This causes theorgans to become ischemic, with nutrient and oxygen flow to these organsbecoming impaired. Given the size of the small and large intestine, asignificant fraction of fluid shunted to the cardiovascular system istaken from this organ. The ischemia within the intestine results in abreakdown of the mucosal barrier lining the lumen of the intestine,allowing the luminal contents to exit the intestine and entercirculation.

Among the biological components leaving the intestine are proteasesrequired for the digestion of food. These proteases include, but are notlimited to, trypsin, chymotrypsin, and elastase. Once in thecardiovascular system, these proteases proceed to act upon circulatingblood proteins, causing these blood proteins to be broken apart intopeptide fragments. Given that these peptides have ACE inhibitoryproperties resulting in the lowering of blood pressure, it would beadvantageous to be able to synthesize these peptides for use as an ACEinhibitor.

FIG. 1 illustrates a schematic 100 of the ACE inhibitor productionmethod, which can be implemented in an exemplary embodiment. In certainembodiments, the initial synthesis step could require obtaining aprotein source. This protein would preferably be of human origin tomaximize the homology between the protein being used and the patientreceiving the peptide fragments, however proteins from animal originscan be used in additional embodiments. A protease is added to either agiven plasma protein or plasma and incubated preferably for up to 24hours at up to 37 degrees Celsius preferably and mixed throughout theincubation. Protease inhibitor is then mixed in to inactivate theprotease.

With regard to albumin, the albumin may be of bovine or chicken origin,being derived from either blood serum or eggs. The purity of the proteinshould be at least 80% to ensure that sufficient substrate is availablefor action by the proteases. The proteases incubated with the proteinsource may be any of those located in the lumen of the intestine, in thecardiovascular circulation, or attached to cell membranes but trypsin,chymotrypsin, or elastase would be preferred. Each protease orcombination or proteases may be incubated for up to 24 hours with theprotein source, with lower incubations needed for optimized substrate toprotease ratios. During incubation the incubation mixture should bestirred, or otherwise perturbed, to facilitate the reaction and thetemperature may be up to 40 degrees Celsius and should preferably be at37 degrees Celsius to increase and optimize the rate of reactionkinetics.

In certain embodiments, the concentration of proteases may be up to thesaturation point of the solvent. Preferable concentrations of plasmaprotein should be in the grams per deciliter range. The solvent itselfmay be, but is not limited to, normal saline, phosphate buffered saline,or any buffered solution. The concentration of proteases may becalculated using a ratio relative to the concentration of the plasmaprotein. The ratio may vary, but preferably should be between 1:10,0000and 1:100 protease concentration to plasma protein concentration. Boththe plasma protein and protease should be fully soluble in the solvent.

Following incubation, the protease activity must be quenched using aprotease inhibitor. “Quenching” refers to removing protease activity byany chemical or mechanical means including, but not limited to,filtration, boiling or protease inhibitor. While the amount of proteaseinhibitor used may be up to one tenth of the amount of protease used, itis preferable for the protease inhibitor to be in a ratio relative toprotease of between 1:10,0000 protease inhibitor to protease to 1:100protease inhibitor to protease. The protease inhibitor should be mixedby stirring, or otherwise perturbing, the solution for up to 24 hours.

After incubation with protease inhibitor, the solution will contain amixture of bioactive ACE inhibitory peptides and inert peptides. Thismixture may be purified to select for the ACE inhibitory peptidesthrough centrifugation that separates the 10 kDa peptides from theremaining peptides. Either following this step or following thequenching of the proteases by proteases inhibitors, the mixture may alsobe sterilized to remove any microbial contaminants.

EXPERIMENTAL DATA In Vitro Experiments

FIGS. 2 through 8 illustrate in vitro ACE inhibition in accordance withexemplary embodiments. Albumin was incubated with either trypsin (aprotease) or chymotrypsin (a protease) or a combination of trypsin andchymotrypsin then the protease or protease was inhibited with a proteaseinhibitor (PI) in a solvent of Phosphate Buffer Saline (PBS). The assayuse is specifically designed to chemically test for ACE inhibition. TheAssay used was the Angiotensin-Converting Enzyme (ACE) Inhibition Assayfrom Dojindo Molecular Technologies, Inc™.

FIGS. 2 and 3 illustrate charts 200, 300 of the average values of animalblood pressure following femoral vein and artery cannulation, inaccordance with an embodiment. The average values displayed are for thetotal course of the surgical experiment. Animal blood pressure of a ratwas allowed to stabilized following femoral vein and artery cannulationfor a period of at least 5 min. Subsequent to each peptide injection,animal blood pressure response was recorded via a pressure transducer.Animal blood pressure values were obtained both for a period prior toany injection and following injection effects to ensure that animalblood pressure was stable prior to each injection and before eachfollowing injection. As a result of these periods, the time span of eachinjection was only a fraction of the total time the animal's bloodpressure was measured. This causes average blood pressure values to behigher than those values measured immediately subsequent to eachinjection.

FIG. 4 illustrates a graph 400 of concentrations of 1:500 Albumin toProtease ratio mixed with other compounds, in accordance with anembodiment. The albumin concentration ratio of 1:500 Albumin+Trypsin(A+T) achieved approximately 55% ACE inhibition. Albumin+Chymotrypsin(A+C) and Albumin+Chymotrypsin+Trypsin (A+C+T) both achievedapproximately 70% ACE inhibition. The control of Albumin+ProteaseInhibitor (A+PI) yielded approximately 30% inhibition.

FIG. 5 illustrates a graph 500 of concentrations of 1:1000 Albumin toProtease ratio mixed with other compounds, in accordance with anembodiment. At an albumin to protease ratio of 1:1000, Albumin+Trypsin(A+T) achieved approximately 45% ACE inhibition. Albumin+Chymotrypsin(A+C) and Albumin+Chymotrypsin+Trypsin (A+C+T) both achievedapproximately 65% ACE inhibition. The control of Albumin +ProteaseInhibitor (A+PI) yielded approximately 15% inhibition. PBS +PI by itselfyielded 20% inhibition. These results demonstrate that combining aprotein source such as albumin with a protease results in a level of ACEinhibition far exceeding that of control. The ACE inhibition produced bythe control is considered noise.

In Vivo Experiments

Sprague-Dawley rats were anaesthetized with isoflurane gas and theirfemoral arteries and veins were used to monitor mean arterial pressureand for peptide injections, respectively. The protocol for producing thepeptides is as follows:

For both experiments, the concentration of albumin was 0.2 g/mL andadded 1% of protease was added. 0.1 g albumin was added in 0.5 mL salinealong with 1mg of protease. The solution was stirred and incubated at 37degrees Celsius for 30 minutes. To stop the reaction and remove theproteases, the incubated solution was centrifuged for 20 minutes in a 3k filter column, which rendered a little more than 0.2 mL offlow-through to inject. Thus, to be consistent, 0.2 mL of each solutionwas injected into the rat so the blood pressure was up and stable againfor the next injection.

FIG. 6 illustrates a graph 600 comparing blood pressure after injectionwith trypsin and albumin, in accordance with an embodiment. A rat wasgiven a peptide injection derived from combining albumin with trypsin.In the graph 600, the blood pressure is tracing immediately followinginjection of the peptides. Peptides generated by combining trypsin andalbumin resulted in a blood pressure decrease of approximately 30 mmHg.

FIG. 7 illustrates a graph 700 comparing blood pressure after injectionwith chymotrypsin and albumin, in accordance with an embodiment. A ratwas given a peptide injection derived from combining albumin withchymotrypsin. In the graph 700 the blood pressure is tracing immediatelyfollowing injection of the peptides. Peptides generated by combiningchymotrypsin and albumin resulted in a blood pressure decrease of 25mmHg.

FIG. 8 illustrates a graph 800 comparing blood pressure after injectionwith chymotrypsin and plasma, in accordance with an embodiment. A ratwas given a peptide injection derived from combining plasma withchymotrypsin. In the graph 800, the blood pressure is tracingimmediately following injection of the peptides. Peptides generated bycombining chymotrypsin and plasma resulted in a blood pressure decreaseof 25 mmHg.

FIGS. 6-8 demonstrated a visible drop in the rat's Mean Arterial BloodPressure (MABP) following each injection. As seen in the graphs in FIGS.6-8 , peptides generated by combining trypsin and albumin resulted in adecrease of approximately 30 mmHg, peptides generated by combiningchymotrypsin and albumin resulted in a decrease of 25 mmHg, and peptidesgenerated by combining chymotrypsin and plasma resulted in a decrease of25 mmHg. The durations of these blood pressure decreases were 1200, 125,and 400 seconds, respectively.

Delivery methods of the disclosed polypeptide angiotensin convertingenzyme (ACE) inhibitor are crucial. Normal drug delivery methods includeIV and oral administration. With reference to oral administration, thepeptides generated by this method are cheaply produced and highly stableand thus may be incorporated in food products. The disclosed peptide ACEinhibitor can be mixed with protein powders, incorporated into bakedgood and put into other food products to provide food products with theadded benefit of lowering a person's blood pressure. Given that peptidesare protein fragments the peptides will not adversely affect taste offood nor will they interact with food ingredients to produce detrimentaleffects. This assertion is supported by the current use of incorporatingprotein into cereals or other food products.

In an embodiment, a method of making polypeptide angiotensin convertingenzyme (ACE) inhibitors from plasma proteins is disclosed. In certainembodiments, the method can comprise mixing a protein source with atleast one protease; quenching the at least one protease mixed with theprotein source to create the polypeptide angiotensin converting enzyme(ACE) inhibitors; and inhibiting the at least one protease mixed withthe protein source completely with the polypeptide angiotensinconverting enzyme (ACE) inhibitors.

In another embodiment, the protein source is mixed with the at least oneprotease for up to 24 hours at up to 40 degrees Celsius. In anembodiment, the protein source is selected from at least one of serumalbumin and macroglobulin. In yet another embodiment, the protein sourceis at a concentration up to a saturation point of the protein source. Inanother embodiment, the at least one protease is selected at least oneof trypsin, chymotrypsin, and elastase. In some embodiments, the atleast one protease is at a concentration up to one tenth of aconcentration of a plasma protein. In yet another embodiment, quenchingcomprises removing protease activity by any chemical or mechanical meanscomprising at least one of filtration, boiling, or utilizing a proteaseinhibitor, wherein the protease inhibitor is at a concentration up toone tenth of a concentration of the at least one protease. In certainembodiments, the method can further comprise incorporating thepolypeptide angiotensin converting enzyme (ACE) inhibitors into foodproducts, wherein the food products can comprise at least one of proteinpowder and a baked good and a user consuming the food productscontaining the polypeptide angiotensin converting enzyme (ACE)inhibitors to lower the blood pressure of the user.

In another embodiment, a method of making polypeptide angiotensinconverting enzyme (ACE) inhibitors from plasma proteins is disclosed. Incertain embodiments, the method can comprise: mixing plasma with atleast one protease; quenching the at least one protease mixed with theplasma to create the polypeptide angiotensin converting enzyme (ACE)inhibitors; and inhibiting the at least one protease mixed with theplasma completely with the polypeptide angiotensin converting enzyme(ACE) inhibitors.

In some embodiments, the plasma is mixed with the at least one proteasefor up to 24 hours at up to 40 degrees Celsius. In other embodiments,the at least one protease is selected from at least one of trypsin,chymotrypsin, and elastase. In yet another embodiment, the at least oneprotease is at a concentration up to 1 g/mL. In an embodiment, quenchingcomprises removing protease activity by any chemical or mechanical meanscomprising at least one of filtration, boiling, or utilizing a proteaseinhibitor, wherein the protease inhibitor is at a concentration up toone tenth of a concentration of the at least one protease. In yetanother embodiment, the method can further comprise incorporating thepolypeptide angiotensin converting enzyme (ACE) inhibitors into foodproducts, wherein the food products can comprise at least one of proteinpowder and a baked good and a user consuming the food productscontaining the polypeptide angiotensin converting enzyme (ACE)inhibitors to lower the blood pressure of the user.

In another embodiment, a method of making polypeptide angiotensinconverting enzyme (ACE) inhibitors from plasma proteins is disclosed. Insome embodiments, the method can comprise: mixing plasma with at leastone protease, wherein the at least one protease comprises chymotrypsin;quenching the at least one protease with the mixed plasma andchymotrypsin to create the polypeptide angiotensin converting enzyme(ACE) inhibitors; and inhibiting chymotrypsin mixed with the plasmacompletely with the polypeptide angiotensin converting enzyme (ACE)inhibitors.

In certain embodiments, the plasma is mixed with chymotrypsin for up to24 hours at up to 40 degrees Celsius. In other embodiments, chymotrypsinis at a concentration up to 1 g/mL. In yet another embodiment, quenchingcomprises removing protease activity by any chemical or mechanical meanscomprising at least one of filtration, boiling, or utilizing a proteaseinhibitor, wherein the protease inhibitor is at a concentration up toone tenth of a concentration of chymotrypsin. In some embodiments, themethod can further comprise incorporating the polypeptide angiotensinconverting enzyme (ACE) inhibitors into food products, wherein the foodproducts can comprise at least one of protein powder and a baked goodand a user consuming the food products containing the polypeptideangiotensin converting enzyme (ACE) inhibitors to lower the bloodpressure of the user.

Based on the foregoing, it can be appreciated that a number ofembodiments, preferred and alternative, are disclosed herein. Thetechniques/embodiments described herein are in no way meant to limit thebreadth of potential applications. It will be appreciated thatvariations of the above-disclosed and other features and functions, oralternatives thereof, may be desirably combined into many otherdifferent systems or applications. Also, it can be appreciated thatvarious presently unforeseen or unanticipated alternatives,modifications, variations or improvements therein may be subsequentlymade by those skilled in the art which are also intended to beencompassed by the following claims.

1. A method of making polypeptide angiotensin converting enzyme (ACE)inhibitors from plasma comprising: mixing the plasma with at least oneserine protease in a ratio of between one ten thousandth and one tenthof the concentration protease concentration to plasma proteinconcentration; stirring the mixture of plasma and trypsin to make thepolypeptide angiotensin converting enzyme (ACE) inhibitors; andquenching the at least one serine protease mixed with the plasma.
 2. Themethod of claim 1, wherein the plasma is mixed with at least one serineprotease for between 0.1 hours to 24 hours.
 3. The method of claim 1,wherein the plasma is mixed with at least one serine protease at 0degrees to 40 degrees Celsius.
 4. The method of claim 1, wherein theplasma comprises serum albumin.
 5. The method of claim 1, whereinquenching comprises contacting the at least one serine protease with aprotease inhibitor.
 6. The method of claim 5, wherein the proteaseinhibitor is at a concentration of between one ten thousandth and onetenth of the concentration of the at least one serine protease.
 7. Themethod of claim 1, wherein the at least one serine protease is trypsin.8. The method of claim 1, wherein the at least one serine protease ischymotrypsin.
 9. The method of claim 1, wherein the at least oneprotease is at a concentration of between 1 μg/mL and 1 g/mL.
 10. Themethod of claim 1 further comprising: incorporating the polypeptideangiotensin converting enzyme (ACE) inhibitors into food product; and auser consuming the food products containing the polypeptide angiotensinconverting enzyme (ACE) inhibitors to lower the blood pressure of theuser.
 11. A method of making polypeptide angiotensin converting enzyme(ACE) inhibitors from blood serum comprising: mixing the blood serumwith chymotrypsin in a ratio of between one ten thousandth and one tenthof the concentration protease concentration to blood serum proteinconcentration; stirring the mixture of blood serum and trypsin to makethe polypeptide angiotensin converting enzyme (ACE) inhibitors; andquenching the at least one serine protease mixed with the blood serum.12. The method of claim 11, wherein the blood serum is mixed with atleast one serine protease for between 0.1 hours to 24 hours.
 13. Themethod of claim 11, wherein the blood serum is mixed with at least oneserine protease at 0 degrees to 40 degrees Celsius.
 14. The method ofclaim 11, wherein the blood serum comprises serum albumin.
 15. Themethod of claim 11, wherein quenching comprises contacting thechymotrypsin with a protease inhibitor.
 16. The method of claim 15,wherein the protease inhibitor is at a concentration of between one tenthousandth and one tenth of the concentration of the chymotrypsin. 17.The method of claim 11, further comprising contacting the blood serumwith trypsin.
 18. The method of claim 11, further comprising contactingthe blood serum with elastase.
 19. The method of claim 11, wherein thechymotrypsin is at a concentration of between 1 μg/mL and 1 g/mL. 20.The method of claim 11 further comprising: incorporating the polypeptideangiotensin converting enzyme (ACE) inhibitors into food product; and auser consuming the food products containing the polypeptide angiotensinconverting enzyme (ACE) inhibitors to lower the blood pressure of theuser.